Process for producing oleandomycin



United States Patent 2,842,481 I PROCESS FOR PRODUCING OLEANDOMYCIN Edwin J. Ratajak, Roslyn, and Robert C. Nubel, Levittown, N. Y., assignors to Chas. Pfizer & Cir, Brooklyn, N. Y., a corporation of Delaware No Drawing. Application March 12, 1957 Serial No. 645,420

sonims. Cl. 195-114 This invention relates to a new and improved method for the production of oleandomycin whereby the yield of product is increased and thexcosts of production are reduced. In particlular, it is concerned with the production of oleandomycin by fermenting a. nutrient me dium with anoleandomycin-producing microorganism of organic dye is. introduced which will cause an increase in the'amount of oleandomycin elaborated. The improve ment of this process comprises conducting the fermentation in the presence of an organic dye chosen from the group consisting of the acid salts of'2, 8-di(dimethylamino) acridine, the acid salts of 2,8-di( dimethylamino) xanthydrol, and a mixture comprising the acid salts of 2,8-diaminoacridine and 2,8-diam ino, 10 methylacridinium chloride.

Oleandomycin is an antibiotic (commercially available from Chas. Pfizer & Co., Inc., under the trademark,

Matromycin) formed during thecultivation under con ganism known as Streptomyces antibioticus. This process is described by B.v A. Sobin, J-. B. vRoutien andiT.

Lees in U. S.'Patent No. 2,757,123, issued on July 31, 1956. It is to be understood that for the. production of oleandomycin the present. invention is not limited to any particular organism, but itis intended to include the use of variants of any new strain of this species as well as Inc.,

trolled conditlons of a strain of aspecles ofmlcroor- 2,842,481 Patented July 8, 1958 ice , ing the organic dye until substantial antibacterial activity after fermentation has commenced.

is imparted to said medium. In particular, this process comprises adding the dye to the fermentation medium, inoculating the medium with a culture of S. antibioticus and permitting growth of this microorganism until a commercially useful. quantity of oleandomycin is thereby produced. Only a small amount of such dye need be present in the fermentation medium, generally in the concentration range of from about 0.005 g. to 2.00 g. per liter. The dye may be added either initially or sometime In general, it is most desirable to add the dye to "the medium within hours of initiation of the fermentation.

The fermentation broth must have present in it an organic dye selected from'the class of acid salts of the organic bases described herein. For this purpose, it is desirable that there be present at least 0.005 g. of dye per liter in the fermentation medium. In particular, the preferred. acid. salt of this series is the hydrochloride. These hydrochlorides are chosen from the group consisting of 2,8-di(dimethylamino) acridine hydrochloride are all found to be extremely valuable for use in the any mutuants produced from the described organism by;

various means, such as X-radiation, ultraviolet radiation, and the like. i I

The. previous invention included the use of a fermentationmedium which gave good yields of oleandomycin, but the improved process of the; present inventionaffords the desired product in greatly increased yields, andthe process is thereby rendered so much more efiicient than, that known in the prior art. that. a. medium which contains the dyes describedherein The data accumulated reveals never has exhibited a potency increase of less than 10% despite the introduction of many experimental variables.-

The organic dyes which are suitable for the process of this invention are acid salts of organic bases selectedifrom mycin medium results in the production of substantial potency increases, an effect that has been repeatedly reprocess of this invention.

The cultivation of the microorganism S. antibioticus preferably takes place in aqueous nutrient media under submerged conditions of agitation and aeration. Nutrient media which are useful for this process include a carbohydrate, such as sugars, starch, glycerol, and corn meal; a source of organic nitrogen, such as casein, soybean meal, wheat gluten, cottonseed meal, lactalbumin and tryptone; a source. of growth substances, such as distillers solubles, yeast extract, molasses fermentation residues, as

Well as mineral salts, such as sodium. chloride, potassium phosphate, sodium, nitrate, magnesium sulfate; trace minerals, such as copper, zinc and iron may also be utilized with desirable results. If excess foaming is encountered during. thefermentation, anti-foaming agents, suchas vegetable. oils, may be added to the fermentation medium. During the fermentation, the pH of the medium tends to remain rather constant in the range of pH 5.5

to 8.0, but to prevent any variations that may occur,

' throughout the transfer of the inoculum and throughout thegrowthof the microorganism. The highest yields are obtained in stainless steel fermentors. The cultivation of the microorganism S. antibioticus preferably takes placev ata temperature in the range of 24 to 30 C. In general, v the fermentation process is most satisfactorily conducted for a time period of from about one to five days, I Y

ilnoculum for the preparation of oleandomycin by the growth of S. antib'ioticus may be obtained by employing growth from slants on such media as Emersons agar or )eef lactose. The growth may be used to inoculate either ihaken flasks or inoculum tanks for submerged growth, )r the inoculum tanks may he seeded from the shaken iasks. The growth of the microorganism usually reaches ts maximum in about two or three days. However, rariations in the-equipment used as well as the rate of teration and agitation may afiect the speed with which maximum activity is attained.

Some of the typical results obtained by the use of recess of this invention are summarized in Table I vherein Acridine Orange is the dye employed:

CABLE I.EFFECT OF ACRIDINE ORANGE IN THE ELABORATION OF OLEANDOMYCIN \cridine Orange, g./l.: Oleandomycin, 'y/ml.

None (control) 255 Hence, the effect produced by the addition of Acridine )range dye to the S. antibioticus fermentation medium vith regard to the elaboration of oleandomycin is readily lemonstrated.

This invention is further illustrated 'by the following :xamples which are not to be considered as imposing lily limitation thereon. 1

Example I A slant of S. antibioticus ATCC 11891 wasicultivated 11 agar under controlled conditions in order to develop pores for the purpose of inoculating a nutrient medium raving the following composition:

' Grams Ierelose (dextrose hydrate) 20 oybean meal l5. )isti'llers solu'bles 5 Zorn meal Tap water, in suflicient amount for a 1000 ml. solution,

adjusted to pH 7.0-7.2 with potassium hydroxide.

lerelose (dextrose hydrate) 60 oybean meal 18 I Iistillers solubles 5 orn meal 12 ap water in suflicient amount for a 1000 ml. total volume, adjusted to pH 7.0-7.2 with potassium hydroxide.

ifter the pH had been adjusted, 5 g. of calcium carbonate ml. of soybean oil antifoam and 0.020 g. of Acridine range dye were added. The mixture was then autolaved at 20 lbs. per sq. in. (250 F.) for 15 minutes in rder to sterilize the contents, before transferring the roth and myceliurn thereto. After seeding the nutrient ledium with the preformed inoculum previously dezribed, the mixture was subjected to agitation and aeraon under aseptic conditions for 72 hours; at 2728 C. )r the first 24 hours, then at 2526 C. for the next 48 ours; during this period, the pH was in the range of .4 to 6.8. Aeration was accomplished by cultivation nder submerged conditions at an air flow rate of one Grams 4 volume of air per volume of medium per minute. After termination of the process, the mycelium was removed by filtration and the filtered broth found to contain 450 of oleandomycin per ml. of solution. This represented a 1.8-fold increase over the control value (255' /rnl.) obtained by the same process wherein the Acridine Orange dye is absent.

' Example II Similar runs were made under the same conditions as those described for Example 1 except that the concentration of Acridine Orange dye was varied in each case within the range of 0.005 to 0.060 g. per liter. Some typical I citrate.

results obtained are reported in Table I. Thus, 0.050 g. of Acridine Orange dye per liter of fermentation medium afforded an oleandomycin potency of 400'y/ml., while 0.025 g. of the same dye yielded an oleandomycin potency of 3607/1111.

a Example III The same procedureas described in Example I was followed here except that Pyronine G was the dye employed; in a similar manner, Acriflav-ine was the dye used. In both cases, it was found that a substantial increase in the amount of oleandomycin elaborated was obtained over that of the control value; in genera-Lwthe order of magnitude of this increase parallels the results obtained in Example 'I.

Example IV The same procedure as described in Example I, II and III was followed here except that various acid salts of the organic 'bases described herein were used in place of the hydrochlorides. For example, the hydrobromide, hydriodi de, phosphate and citrate of 2,8-di(dimethylamino) acridine, 2,-8-di(dimethylamino) xanthydrol, and a mixture comprising 2,8-diaminoacridine and 2,8-diamino-10-methy1acridin-ium chloride, respectively, were all employed, and the results obtained were in substantial agreement with the data previously accumulated.

What is claimed is:

1. In a process for the production of oleandomycin by fermenting a nutrient medium with a culture of Streptomyces antibioticus, the improvement which comprises adding to the medium within 30 hours of initiation of the fermentation an organic dye chosen from the group consisting of the acid salts of 2,8-di( dimethylamino) acridine, the acid salts of 2,8-di (dimethylamino) -xanthydrol,and a mixture comprising the acid salts of 2,8-diaminoacridine and 2,8-diamino-l0-methylacridinium chloride.

2. A process as claimed in claim 1 wherein the acid salts are selected from the class consisting of the hydrochloride, hydrobromide, hydriodide, phosphate and 3. A process as claimed in claim 1 wherein the organic dye is an acid salt of 2,8-di(dimethylamino) acridine.

4.'A process as claimed in claim 1 wherein the organic dye is an acid salt of 2,8-di(dim ethylamino) xanthydrol.

5. A process as claimed in claim 1 wherein the organic dye is a mixture comprising the acid salts of 2,8-diaminoacridine and 2,8-diamino-10-methylacridinium chloride.

6. A process as claimed in claim 1 wherein the said dye is present in the concentration range of from about 0.005 :g. to 2.000 g. per liter of fermentation medium and the process is conducted in the pH range of 5.5 to 8.0 at a temperature in the range of 24 to 30 C. for a time period of from about one to live days.

Wiley'& Sons, Inc., page328. H 

1. IN A PROCESS FOR THE PRODUCTION OF OLEANDOMYCIN BY FERMENTING A NUTRIENT MEDIUM WITH A CULTURE OF STREPTOMYCES ANTIBIOTICUS, AND IMPROVEMENT WHICH COMPRISES ADDING TO THE MEDIUM WITHIN 30 HOURS OF INITIATION OF THE FERMENTATION AN ORGANIC DYE CHOSEN FROM THE GROUP CONSISTING OF THE ACID SALTS OF 2,8-DI(DIMETHYLAMINO) ACRIDINE, THE ACID SALTS OF 2,8-DI(DIMETHYLAMINO) XANTHYDROL, AND A MIXTURE COMPRISING THE ACID SALTS OF 2,8-DIAMINOACRIDINE AND 2,8-DIAMINO-10-METHYLACRIDINIUM CHLORIDE. 